The overall goal of this proposal is to assess protein expression in brain regions from Ts65Dn mice [an animal model of Down syndrome (DS] using two dimensional gel electrophoresis/mass spectrometry (2DGE-MS). 2DgE-MS is necessary because altered mRNA expression does not always correlate with altered protein levels. A single gene frequently gives rise to multiple protein isoforms by posttranscriptional mechanisms, such as alternative splicing or posttranslational modifications. This effort will result in improved analytical methods, specifically 2DGE-MS quantitation and enhancement of the range of proteins analyzed by this approach. The method of choice will be difference gel electrophoresis (DGE) followed by MS analysis of the separated proteins. For proteins that are difficult to separate by 2DGE-MS, complementary approaches include fractionation on 1-dimensional SDS gels, slicing of the gels, proteolysis of the slices, and analysis by LC/MS/MS. For small proteins and peptides, identification will be done without proteolysis using known standards using our previously published procedures. We will compare the hippocampus and cerebellum of Ts65Dn mice and littermate normal controls at 3, 6 and 12 months of age because they span ages in which behavioral and histological changes occur in the Ts65Dn mice. We will initially focus on unfractionated extracts and mitochondria. Unfractionated extracts will be examined because of the richness of protein representation. Mitochondria will be analyzed because the mitochondrial proteome is relatively small, and because altered mitochondrial metabolism, including generation of reactive oxygen species, is hypothesized to be abnormal in DS. We hypothesize that: 1) the protein levels of many proteins, encoded not only on chromosome 21 (mouse chromosome 16) but throughout the genome, will be altered in the Ts65Dn mouse model of DS and therefore most probably in DS as well; 2) levels of proteins in the same biological (e.g., biochemical, signaling, developmental) pathway are coordinately regulated whether or not their genes are trisomic, 3) compensation for trisomy can occur via reduction of the levels of the proteins(s) encoded by the trisomic gene(s) or via reduction of the levels of other proteins in the biological pathway; 4) 2DGE-MS can identify coordinately regulated proteins, resulting in the identification of new biological pathways important for the phenotype of DS; 5) alternative processing of mRNAs commonly results in many protein isoforms from the same gene, greatly enhancing the complexity of the proteome; and 6) the mitochondrial proteome is altered in DS in ways that reflect altered mitochondrial metabolism in DS, contributing to the disabilities associated with DS. Understanding of the alterations in protein levels in the Ts65Dn mouse model of DS should lead to new approaches of ameliorate the cognitive and behavioral aspects of DS.